Detection of Macrolide-Resistant Streptococcus pneumoniae Genes and Its Clinical Outcomes in a Tertiary Teaching Hospital in Malaysia

Background Streptococcus pneumoniae is one of the leading causes of mortality and morbidity worldwide. The dramatic increase in in-vitro resistance of antimicrobial agents, particularly beta-lactams and macrolides, makes pneumococcal infections difficult to treat. The aim of this study was to describe the drug resistance rate, assess the prevalence of macrolide-resistant genes and review the clinical complications of pneumococcal infections among patients presented to Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia. Methods This is a descriptive cross-sectional study. All S. pneumoniae isolates collected from clinical specimens within a 1-year period were subjected to selected antimicrobial susceptibility testing using E-test strips. Polymerase chain reaction (PCR) analysis was conducted to detect macrolide-resistant determinants. The patient’s clinical data were obtained from clinical notes. Results A total of 113 patients with a positive growth of S. pneumoniae were included in the study. The most common predisposing factors among them were bronchopulmonary diseases (15.9%). The penicillin-resistant rate was 7.1%, with minimal inhibitory concentration (MIC) ranging between 0.012 μg/mL and >32 μg/mL, and the erythromycin-resistant rate was 26.5%, with a MIC range of 0.03 μg/mL–> 256 μg/mL. Most of the erythromycin-resistant isolates were found to have the mef(A) gene (50.4%) and the erm(B) gene (20%); 16.7% had a combination of genes mef(A) and erm(B), and 13.3% had none of the two genes. Community-acquired pneumonia is the predominant type of pneumococcal infection. There was no significant association between the presence of macrolide resistance determinants and mortality (P = 0.837) or complications (P > 0.999 for empyema and cardiac complication; P = 0.135 for subdural abscess). Conclusion The majority of erythromycin-resistant isolates were found to have the mef(A) gene, followed by the erm(B) gene and a combination of genes mef(A) and erm(B).


Introduction
Streptococcus pneumoniae is one of the leading causes of mortality and morbidity worldwide, posing a major public health problem.It causes more serious infections in infants under 2 years old (1) and adults older than 65 years old (2).S. pneumoniae is a Grampositive bacteria spread by airborne droplets, and it is a primary cause of bacterial pneumonia, acute otitis media, sinusitis, bacteremia and meningitis.The World Health Organization (WHO) estimates that, in the Asia Pacific region, 49 out of 98 cases of pneumonia deaths in children are due to pneumococcal pneumonia.Several studies reported that pneumonia is the most common pneumococcal disease in Malaysia (3,4).Additionally, about 4% of the 7,000 deaths among children less than 5 years old were thought to be caused by S. pneumoniae (5).
The dramatic increase in the prevalence of antibiotic resistance, especially against firstline antibiotics such as penicillin and macrolide, makes pneumococcal infections difficult to treat, especially in children and the elderly (6).In some Asian countries, the reported prevalence of penicillin and macrolide resistance was the highest in the world (7,8).Previous surveillance studies showed that more than 60% of pneumococcal isolates from Asian countries were resistant to erythromycin and 22.7% were penicillin-resistant (9).In China, erythromycin resistance rates for S. pneumoniae increased from 35% to 53% in 1996-1999, to over 75% by 2001, and South Korean resistance rates against erythromycin remained high from 1996 to 2001 (75%-85%).While for Malaysia, 32% were resistant to erythromycin in the year 2012 (9).
Ribosomal methylation encoded by erm(B) gene was the most common mechanism of erythromycin resistance in China, Taiwan, Sri Lanka and Korea (7).While in Hong Kong, Singapore, Thailand and Malaysia, an efflux pump encoded by the mef(A) gene was more common.Erm(B) gene was found in more than 50% of pneumococcal isolates either alone or in combination with mef(A) gene in most Asian countries except Hong Kong, Malaysia and Singapore (7).Although antibiotic resistance among S. pneumoniae has been increasingly noted worldwide, the clinical significance of invitro resistance is not widely studied (10,11).The aim of this study is to describe the antibiotic susceptibility pattern and the distribution of macrolide-resistance determinants of S. pneumoniae isolated from patients presented to Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia.

Pneumococcal Isolates
A total of 113 non-duplicate isolates of S. pneumoniae were collected from patients admitted to HUSM from June 2014 to December 2015.Pneumococcal isolates were collected from various clinical specimens including sputum, throat swab, endotracheal secretion, nasopharyngeal swab/aspirate, blood, cerebrospinal fluid (CSF), pus swab, high vaginal swab (HVS) and other body fluids.The isolates were presumptively identified as S. pneumoniae by their colony morphology, Gram-stain results and susceptibility to 5 µg optochin disc (ethyl hydrocuprein hydrochloride, Becton, Dickinson and Company, USA).The isolates were stored at -70 o C in brain heart infusion broth plus 20% glycerol in the laboratory until further testing.

Antimicrobial Susceptibility Test
The minimal inhibitory concentrations (MICs) of six antimicrobial agents: i) penicillin, ii) erythromycin, iii) azithromycin, iv) amoxicillin-clavulanic acid, v) trimethoprimsulphamethoxazole and vi) vancomycin were determined by using E-test strips (BioMerieux SA, France) according to manufacturer's recommendations.The MIC results are interpreted according to Clinical and Laboratory Standard Institute (CLSI) guidelines, 2015.S. pneumoniae ATCC 49619 was used as the control strain.

Detection of Macrolide-Resistant Genes; erm(B) and mef(A) Genes
Out of 113 isolates of S. pneumoniae, 108 were subjected to polymerase chain reaction (PCR) analysis to determine the presence of macrolide-resistant genes.Three of the isolates were non-viable and the other two isolates were contaminated with other bacteria.According to the manufacturer's instructions, DNA extractions were performed using a DNA extraction kit (DNeasy Blood & Tissue Kit, Qiagen, USA).The PCR mixture consists of a 20 µL total volume containing extracted DNA template, DreamTaq DNA polymerase (Thermo Scientific, Malaysia), 10X DreamTaq buffer which contains 20 mM MgCl2, 2 mM dNTP Mix, nuclease-free water and primer mixture.The primer sequences for erm(B) gene are 5'-CGTACCTTGGATATTCACCG-3' and 5'-GTAAACAGTTGACGATATTCTCG-3' whilst for mef(A) gene are 5'-CTGTATGGAGCTACCTGTCTGG-3' and 5'-CCCAGCTTAGGTATACGTAC-3' (12).The primer mixture was prepared by mixing an equal concentration of genes erm(B) and mef(A) primers (20 pmol/µL) for each forward and reverse primer.The amplification was performed with a thermal cycler (MJ Research Peltier Thermal Cycler, GMI, USA).The positive control strains; S. pneumoniae ATCC 700673 (Hungary19A-6) for erm(B) gene and S. pneumoniae ATCC 51916 (Tennessee23F-4) for mef(A) gene (13) and a negative control were included in each run.Following amplification, 2 µL of the PCR products were electrophoresed on 1% agarose gel and visualised using a UV light transilluminator (Syngene, USA) with GeneSnap image analysis software.

Statistical Analysis
Statistical analysis was performed using SPSS, version 22.0.Fisher's exact test or X 2 test was applied to determine the association between the presence of macrolide resistance genes with mortality and complications.

Demographic and Clinical Characteristics of Patients with S. pneumoniae Infection
Among the 113 patients, 22 (19.5%) were children aged 5 years old or below and 16 (14.2%)were elderly patients (≥ 65 years old).The median age of the patients was 33 years old.Of these patients, 73 (64.6%) were males and 40 (35.4%) were females.Community-acquired pneumonia was the most common type of infection (59.3%), followed by conjunctivitis (8.8%) and bacteremia (7.1%).About 9.9% of the patients had S. pneumoniae (the majority were from ETT specimens).The most common predisposing factors among patients with culture-positive S. pneumoniae were bronchopulmonary diseases (15.9%), underlying malignancy (6.2%) and chronic renal disease (4.4%).

In vitro Susceptibility Pattern of S. pneumoniae Isolates
The susceptibility pattern of 113 S. pneumoniae isolates was shown in Table 1 and the MIC distribution of macrolide antibiotic based on the presence of macrolide resistance determinants was shown in Table 3.

Distribution of erm(B) and mef(A) Genes
Out of 108 isolates subjected to PCR analysis, erm(B) gene, mef(A) gene or a combination of erm(B) and mef(A) genes were identified in 31 (27.4%)isolates.Of these isolates, 20 (64.5%) had mef(A) gene alone and 6 (19.4%) had erm(B) gene alone, whereas 5 (16.15) contained both mef(A) and erm(B) genes.The PCR gel image showed amplified DNA fragments, as demonstrated in Figure 1.

Impact of Resistance to Mortality and Complications
Six patients (5.3%) died in the hospital while receiving treatment.One patient (0.9%) had empyema-related complications and another had subdural abscess-related complications.Two (1.8%) developed arrhythmia.The results (Tables 4 and 5) showed that there is no significant association between mortality rate and the development of complications between patients harbouring macrolide-resistance determinants with those without macrolideresistance determinants.

Discussion
In Malaysia, the prevalence of macrolideresistant S. pneumoniae appeared to have increased over the years from 36.8% in 2001 (14) to 46.2% in 2005, then drastically increased to 62.2% in the year 2010 (15,16).In our study, the rates of susceptibility towards macrolide antibiotics were not much different to those of a  previous study (16), where the resistance rates to erythromycin and azithromycin were 26.5% and 30.1%, respectively.However, we reported a decrease in erythromycin resistance compared to more recent studies.However, the resistance rate towards erythromycin in 2010, which was more than 60%, was higher (14,15).The level of macrolide resistance has increased remarkably, with very high MIC 90 s value (64 µg/mL to ≥ 128 µg/mL) in Asian countries, including Malaysia (9).In this study, we found that isolates which are resistant to erythromycin or azithromycin also have high MIC values.The majority of them have an MIC of > 256 µg/mL, with MIC 90 values of 32 µg/mL and 128 µg/mL for erythromycin and azithromycin, respectively.Compared with previous studies that investigated S. pneumoniae isolates in the years 2008-2010, the MIC 90 s of erythromycin are higher, with MIC values of ≥ 256 µg/mL (14,15).In this study, 96.7% of erythromycin-resistant isolates were also resistant to azithromycin, which is consistent with previous studies where 94.4%-97% of the erythromycin-resistant pneumococcal isolates had concomitant erythromycin and azithromycin resistance (6,15).
The erm(B) gene-mediated resistance in pneumococci gives high-level resistance (MLS B phenotype), with MIC values typically ≥ 64 µg/mL.In contrast, the efflux pump (M phenotype) encoded by the mef(A) gene confers a low-level resistance, with MIC usually 1 µg/mL-32 µg/mL (9,17,18).Similarly, in this study, among the 30 erythromycin-resistant pneumococci, isolates that carry the erm(B) gene alone or both erm(B) and mef(A) genes showed higher MICs, ranging from 2 µg/mL to > 256 µg/mL, with MIC 90 values for erm(B) gene being > 256 µg/mL.This is in contrast to isolates carrying mef(A) gene alone, which have much lower MICs ranging from 1 µg/mL to 32 µg/mL and a MIC 90 of 4 µg/mL for mef(A) gene.These findings were in accordance with the other study that reported erythromycin-resistant S. pneumoniae isolates with both erm(B) and mef(A) genes showed very high MICs ≥ 256 µg/ mL (14).
Ones risk of contracting pneumococcal infections varies depending on the age and gender of the individual.The prevalence of pneumococcal disease is substantially higher in children and the elderly above 65 years old than in young adults (2).The study's findings also emphasise on the preponderance of pneumococcal disease among males, which might be linked to the higher incidence of underlying factors such as smoking and exposure to outdoor air pollution among males.Deficiencies in the non-specific or specific defence mechanisms against colonisation, aspiration or invasion of S. pneumoniae increase the likelihood of pneumococcal disease among this group of patients.
The clinical significance of the in-vitro resistance of macrolide antibiotics is still controversial (19).The failure of macrolide therapy caused by erythromycin-resistant strain in patients with pneumococcal diseases has been reported in previous studies (18,20).A study investigating the mortality rates associated with invasive pneumococcal diseases reported almost similar findings with patients with erythromycin-resistant (18%) or erythromycin-susceptible strains (14%) (21).This study also had similar findings, where the mortality rate among patients with macrolideresistant strains was not significantly different from that of patients with macrolide-susceptible strains (P > 0.999 for erythromycin and P = 0.667 for azithromycin).In this study, we also specifically assessed the relationship between the macrolide resistance determinants and the mortality and complications in patients with pneumococcal diseases since these resistant determinants are the main cause of macrolide resistance.This revealed a similar result where mortality and complication in patients carrying the resistance determinants did not differ from those in patients without resistance determinants.These findings were supported by another study by the Asian Network for Surveillance of Resistant Pathogens (ANSORP) group that reported that pneumococcal infection with erythromycin-resistant strains was not significantly associated with more severe illness or higher mortality (22).
The prevalence of the macrolide-resistant determinant gene of S. pneumoniae can provide the management teams in hospitals with crucial insights for making appropriate decisions on antimicrobial usage in patients' treatment.By knowing its prevalence, healthcare professionals can understand the likelihood of resistance and choose alternative antibiotics for patients with macrolide-resistant infections, improving treatment efficacy.They may avoid unnecessary macrolide prescriptions in areas with high resistance rates, reducing the risk of further resistance development and preserving the effectiveness of these antibiotics.Furthermore, they can implement targeted infection control measures to limit the spread of macrolideresistant infections within healthcare facilities.
This study has several limitations.The identification of S. pneumoniae isolates based on colony morphology, Gram-stain results and susceptibility to the optochin disc reflects the use of classical microbiological methods that have been historically relied upon for bacterial identification.These methods offer a costeffective and relatively simple way to distinguish between different streptococcal species which is often encountered in clinical settings.However, the challenge arises because closely related species like S. pseudopneumoniae and S. mitis share similar characteristics and can be mistaken for S. pneumoniae using these conventional techniques.
The similarity in optochin susceptibility and bile solubility between these species can lead to misclassification.As advancements in molecular techniques and genetic analysis have emerged, it has become apparent that incorporating more precise and specific methods, such as DNA sequencing and PCR-based assays, can enhance the accuracy of S. pneumoniae identification.However, due to limited resources and a small amount of grant money allocation, the molecular identification method could not be used.
Another limitation was the inadequacy of clinical data collected from medical records that largely depend on the clinician's observation and interpretation.Incomplete documentation of the medical records will largely influence our data, especially on the predisposing factors and outcome of the diseases.Finally, since the sources of the isolates were collected only from the state of Kelantan, data from this study may not be representative of the Malaysian population.

Conclusion
The majority of erythromycin-resistant isolates were found to have a mef(A) gene, an erm(B) gene or a combination of mef(A) and erm(B) genes.Overall, the isolates showed good susceptibility towards all antibiotics tested except for azithromycin.The outcome and complications of pneumococcal diseases were not significantly different between macrolideresistant groups and macrolide-susceptible groups.They also were not affected by the presence of macrolide-resistant determinants in the pneumococcal isolates.Current data regarding the in-vitro susceptibility patterns of S. pneumoniae and the prevalence of the macrolide-resistant determinants gene could help the management team make appropriate decisions regarding antimicrobial usage in treating patients.Continuous surveillance of antibiotic resistance at the national level is very important to monitor the changing trends in antimicrobial resistance in Malaysia.

Table 1 .
Susceptibility rates of S. pneumoniae isolate for six antimicrobial agents (n = 113)

Table 5 .
Association between macrolide resistance determinants with complications (n = 111) Notes: a Fisher's exact test was applied; NV/C= non-viable/contaminated

Table 4 .
Association between macrolide resistance determinants with clinical outcome (n = 111)